Ultraviolet radiation (UVR) is a well-established carcinogen and leading environmental risk factor for melanoma, one of the most aggressive and treatment-resistant malignancies. Melanocytes are vulnerable to accumulation of UV-induced mutations that can lead to malignant transformation if unchecked by immunosurveillance. However, continued survival of melanocytes is essential for photoprotection from future UV insults, suggesting that melanocytes may require enhanced immune tolerance. Vitiligo is caused by failure of tolerance in which autoimmunity against melanocytes results in depigmented patches of skin. Thus melanoma and vitiligo reflect the extremes of inadequate or excess immune activity against the melanocytic lineage. This project aims to develop mouse models for two main objectives: (1) to investigate one pathway by which UVR may promote melanocyte-mediated immune tolerance, and (2) to evaluate the role of UV-induced neoantigens and epitope spread in immune responses against melanomas. Ultimately we hope to use this knowledge to suggest approaches for preventing vitiligo, enhancing anti-melanoma immunity, and increasing the proportion of melanoma patients who benefit from immunotherapy. MITF is the master transcriptional regulator of the melanocytic lineage and is induced by UVR as part of the tanning response. Preliminary data suggest that PD-L1, a T cell coinhibitory signaling molecule, is a transcriptional target of MITF. In the first aim, mechanisms of PD-L1 regulation by MITF in both the absence and presence of UVR will be further characterized in vitro and in mice. Next, rates of vitiligo development in UV-irradiated mice with or without conditional PD-L1 knockout in melanocytes will be compared to determine whether PD-L1 expression is required for immune tolerance to UV-induced neoantigens. Lastly, to assess the role of the MITF-PD-L1 axis in melanoma-mediated immune evasion, immune responses against melanoma cells following disruption of MITF- or IRF1-binding sites in the PD-L1 enhancer by CRISPR will be compared in a syngeneic tumor graft mouse model. The goal of the second aim is to understand the effects of increasing neoantigen loads on immune responses in melanoma. To pursue this aim, single cell clones of C57BL/6 congenic mouse melanoma cells with increasing numbers of UV-induced mutations will be generated and characterized. Next, these clones will be grafted into C57BL/6 mice that will be treated with immune checkpoint inhibitors to break immune tolerance. The rates of tumor growth and survival and magnitude of immune responses against melanoma clones with different neoantigen loads will be compared. The extent of epitope spread in these mice will also be studied, as clinical trials have suggested that immune checkpoint inhibitors potentiate epitope spread of immune activity against initial melanoma-specific epitopes to normal melanocytic antigens. To investigate this question, rates of vitiligo development in mice engrafted with melanomas containing different neoantigen loads will be compared.
Metastatic melanoma is a deadly disease with an expected five-year survival rate of only 16%, and melanoma incidence is rising among both men and women in the United States. Vitiligo, the most common pigmentation disease, can be disfiguring and often psychologically devastating. The goal of this project is to better understand the effects of ultraviolet light exposure on immune responses to melanocytes and melanomas, in the hope that this knowledge will suggest approaches for preventing vitiligo and developing better melanoma immunotherapies.
|Lo, Jennifer A; Fisher, David E; Flaherty, Keith T (2015) Prognostic Significance of Cutaneous Adverse Events Associated With Pembrolizumab Therapy. JAMA Oncol 1:1340-1|